Methods for human allografting

ABSTRACT

A method of preparing a human recipient for a graft from a human which includes: administering donor peripheral blood progenitor cells to the recipient, and providing a minimally ablative.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/118,894, filed on Feb. 4, 1999, the contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The invention relates to tissue and organ transplantation.

SUMMARY OF THE INVENTION

In another aspect, the invention features a method of preparing arecipient, e.g., a human patient, for a graft from a donor mammal of thesame species. The method includes:

Creating hematopoietic space in the recipient, e.g., by administering tothe recipient an amount of irradiation or chemotherapy sufficient toallow the induction of mixed chimerism in the recipient. The irradiationshould be administered in two (or more) separate doses, preferably equaldoses. By way of example, 300 cGy of whole body irradiation can beadministered as 150 cGy on each of two successive days of whole bodyirradiation. The irradiation step can be replaced by chemotherapydescribed below;

Administering to the recipient one or more chemotherapeutic agents,e.g., cyclophosphamide, busulfan, fludararabine, or any combinationthereof. The chemotherapeutic agent can be a radiomimetic (i.e. an agentthat interacts with DNA in a mechanism similar to ionizing radiation) ora non-radiomimetic. In one embodiment, a single-agent chemotherapeuticregimen is administered. For example, cyclophosphamide can beadministered (preferably, intravenously) at a dose of 50 mg/kg/day forthree consecutive days (e.g., on days −5, −4 and −3 prior toadministration of stem cells or bone marrow on day 0). Alternatively, acombination of agents, e.g., cyclophosphamide, busulfan and/orfludararabine, can be used. Exemplary combinations includecyclophosphamide and fludararabine, and cyclophosphamide and busulfan.Analogues of these compounds may also be incorporated into the finalregimen, e.g., substituting treosulfan for busulfan; purine ornucleoside analogs of these agents, e.g., fludarabine. The combinationof agents can be administered sequentially or, preferably, concurrently.Preferably, the cyclophosphamide is administered intravenously.

Creating thymic space in the recipient, e.g., by administering to therecipient, an amount of thymic irradiation sufficient to kill orotherwise inactivate recipient thymocytes. By way of example, 700 cGy ofthymic irradiation can be administered to the recipient. This step canbe replaced with other methods of creating thymic space, describedbelow;

Administering to the recipient, one or more doses of at least oneantibody which depletes T cells. The antibodies can be administeredbefore, on, or after, the day of graft implantation. In a preferredembodiment the antibodies are administered up to or on the day of graftimplantation. Suitable antibodies include, anti-CD4, anti-CD8, andanti-CD2 antibodies. Anti-CD2 antibodies are preferred. For example, themethod can include, administering to the recipient, an anti-CD2monoclonal antibody, e.g., administering to the recipient, at least onedose, and possibly two, three or four, doses of an anti-CD2 monoclonalantibody, e.g., before or up to the day of graft implantation. By way ofexample, four doses of an anti-CD2 monoclonal antibody, e.g., MEDI-507,can be administered. Optionally, the doses should be more or less evenlyspaced. The last can occur the day before transplantation, e.g., dosesadministered on days −4, −3, −2, −1 (wherein day 0 is the day of grafttransplant and day −1 is the day prior to day 0). A dose of 0.1 mg/kg onday −4 and of 1.0 mg/kg on days −3, −2, and −1, of the anti-CD2monoclonal antibody MEDI-507 has been found to be effective.Alternatively, the antibody may be administering up to several weeksprior to the graft implantation. As one example, a dose of 0.1 mg/kg canbe administered on day −7, and a single dose of 0.5 mg/kg can beadministering on day −6. Analogous doses of other anti-CD2 antibodiescan be used;

Optionally, administering to the recipient, a treatment which reducesthe side effects on the administration of anti-T cell antibodies, e.g.,administering an agent which suppresses the release of antibody-inducedcytokine release. By way of example, the method can includeadministering to the recipient, a steroid immunosuppressant, e.g.,methylprednisolone. The immunosuppressant can be administered prior toone or more, e.g., prior to each, or prior to the first, administrationof a T cell depleting antibody, e.g., an anti-CD2 antibody. By way ofexample, an intravenous dose of methylprednisolone sodium succinate, 8mg/kg, to a maximum dose of 500 mg has been found to be effective. Themethylprednisolone can be administered 1-4 hours (preferably, between 1and 2 hours) before antibody administration

Optionally, administering a treatment which inhibits or depletes splenicT cells and/or B cells. Examples of such treatment include theadministration of anti-T cell antibodies, anti-B cell antibodies orimmunosuppressive agents, in sufficient dosage to inhibit or depletesplenic antibodies, or performing a splenectomy. In a preferredembodiment the method includes performing a splenectomy on therecipient, e.g., on the day of transplantation day 0. Preferably, therecipient is not splenectomized when an anti-CD40 ligand isadministered;

Administering to the recipient, donor hematopoietic progenitor cells,e.g., donor hematopoietic bone marrow cells. Mobilized donor peripheralblood progenitor cells are preferred. Bone marrow can also be used as asource of cells. Hematopoietic stem cells can be administered before,after, or on the day of transplantation (day 0); and

Administering to the recipient, a non-chronic course of animmunosuppressant, e.g., cyclosporine or FK506. By way of example, animmunouppressant can be administered from the day of transplant butdiscontinued after stable chimerism has been established. The dose of animmunosuppressant can be given in two phases, a first phase in which achosen level of immunosuppressant is established, e.g., a levelsufficient to inhibit graft-reactive T cells, followed by a phase inwhich the dose is tapered. E.g., cyclosporine can be administered toprovide a trough whole blood concentration of 400-500 ng/mL, for 35days, then tapered over 7 days and discontinued on Day 42).

In preferred embodiments, the method includes the step of introducinginto the recipient, a graft obtained from the donor. The graft can beother than hematopoietic stem cells or bone marrow, e.g., a heart,pancreas, liver, or kidney.

In a preferred embodiment the donor is related to the recipient, e.g.,is a sibling, parent, or cousin.

In a preferred embodiment the donor is not related to the recipient.

In a preferred embodiment the graft is from a cadaver.

In a preferred embodiment the method includes:

administering to a human recipient, 300 cGy (150 cGy on each of twosuccessive days) of whole body irradiation;

administering to the recipient, 700 cGy of thymic irradiation;

administering a T cell depleting antibody, e.g., an anti-CD2 monoclonalantibody, e.g., administering an anti-CD2 monoclonal antibody, e.g.,MEDI-507, on days −4, −3, −2, −1;

optionally, administering a steroid immunosuppressant, e.g., prior tothe first, or each, antibody administration, e.g., administering a doseof methylprednisolone sodium succinate, prior to the first, or each,antibody administration;

optionally, performing a splenectomy on the recipient, e.g., on the dayof transplantation day 0;

administering to the recipient donor peripheral blood progenitor cells,e.g., on the day of transplantation day 0; and

administering to the recipient a non-chronic course of animmunosuppressant, e.g., cyclosporine started on the day prior totransplantation. By way of example, starting on the day aftertransplantation (Day 1), cyclosporine can be administered to provide atrough whole blood concentration of 400-500 ng/mL, for 35 days, thentapered over 7 days and discontinued on Day 42).

In another aspect, the invention features a method of preparing arecipient, e.g., a human patient, for a graft from a donor mammal of thesame species, without the administration of whole body irradiation. Themethod includes:

Creating thymic space in the recipient, e.g., by administeringadministering to the recipient, an amount of thymic irradiationsufficient to kill or otherwise inactivate recipient thymocytes. By wayof example 1,000 cGy of thymic irradiation can be administered to therecipient. This step can be replaced with other methods of creatingthymic space, as is described below: Administering to the recipient, oneor more doses of at least one antibody which depletes T cells. Theantibodies can be administered before, on, or after, the day of graftimplantation. In a preferred embodiment the antibodies are administeredup to or on the day of graft implantation. Suitable antibodies include,anti-CD4, anti-CD8, and anti-CD2 antibodies. Anti-CD2 antibodies arepreferred. For example, the method can include, administering to therecipient, an anti-CD2 monoclonal antibody, e.g., administering to therecipient, at least two, three or four, doses of an anti-CD2 monoclonalantibody, e.g., before or up to the day of graft implantation. By way ofexample, four doses of an anti-CD2 monoclonal antibody, e.g., MEDI-507,can be administered. The doses should be more or less evenly spaced withthe last occurring the day before transplantation, e.g., dosesadministered on days −4, −3, −2, −1. A dose of 0.1 mg/kg on day −4 andof 1.0 mg/kg on days −3, −2, and −1, of the anti-CD2 monoclonal antibodyMEDI-507 has been found to be effective. Analogous doses of otheranti-CD2 antibodies can be used;

Optionally, administering to the recipient, a treatment which reducesthe side effects on the administration of anti-T cell antibodies, e.g.,administering an agent which suppresses the release of antibody-inducedcytokine release. By way of example, the method can includeadministering to the recipient, a steroid immunosuppressant, e.g.,methylprednisolone. The immunosuppressant can be administered prior toone or more, e.g., prior to each, or prior to the first, administrationof a T cell depleting antibody, e.g., an anti-CD2 antibody. By way ofexample, an intravenous dose of methylprednisolone sodium succinate, 8mg/kg, to a maximum dose of 500 mg has been found to be effective. Themethylprednisolone can be administered 1-4 hours (preferably, between 1and 2 hours) before antibody administration;

Optionally, administering a treatment which inhibits or depletes splenicT cells and/or B cells. Examples of such treatment include theadministration of anti-T cell antibodies, anti-B cell antibodies orimmunosuppressive agents, in sufficient dosage to inhibit or depletesplenic antibodies, or performing a splenectomy. In a preferredembodiment the method includes performing a splenectomy on therecipient, e.g., on the day of transplantation day 0; Administering tothe recipient, donor hematopoietic progenitor cells, e.g., donorhematopoietic bone marrow cells. Mobilized donor peripheral bloodprogenitor cells are preferred. Hematopoietic stem cells can beadministered before, after, or on the day of transplantation (day 0). Anamount of donor hematopoietic stem cells sufficient to result in mixedchimerism in the absence of whole body irradiation is administered; and

Administering to the recipient, a non-chronic course of animmunosuppressant, e.g., cyclosporine or FK506. By way of example, animmunouppressant can be administered from the day of transplant butdiscontinued after stable chimerism has been established. The dose of animmunosuppressant can be given in two phases, a first phase in which achosen level of immunosuppressant is established, e.g., a levelsufficient to inhibit graft-reactive T cells, followed by a phase inwhich the dose is tapered. E.g., cyclosporine can be administered toprovide a trough whole blood concentration of 400-500 ng/mL, for 35days, then tapered over 7 days and discontinued on Day 42).

In preferred embodiments, the method includes the step of introducinginto the recipient, a graft obtained from the donor. The graft can beother than hematopoietic stem cells or bone marrow, e.g., a heart,pancreas, liver, or kidney.

In a preferred embodiment the donor is related to the recipient, e.g.,is a sibling, parent, or cousin.

In a preferred embodiment the donor is not related to the recipient.

In a preferred embodiment the graft is from a cadaver or a living donor.

In a preferred embodiment the method includes:

administering to a human recipient, 1,000 cGy of thymic irradiation;

administering a T cell depleting antibody, e.g., an anti-CD2 monoclonalantibody, e.g., administering an anti-CD2 monoclonal antibody, e.g.,MEDI-507, on days −4, −3, −2, −1;

optionally, administering a steroid immunosuppressant, e.g., prior tothe first, or each, antibody administration, e.g., administering a doseof methylprednisolone sodium succinate, prior to the first, or each,antibody administration;

optionally, performing a splenectomy on the recipient, e.g., on the dayof transplantation day 0;

administering to the recipient donor peripheral blood progenitor cells,e.g., on the day of transplantation day 0; and

administering to the recipient a non-chronic course of animmunosuppressant, e.g., cyclosporine started on the day prior totransplantation. By way of example, starting on the day aftertransplantation (Day 1), cyclosporine can be administered to provide atrough whole blood concentration of 400-500 ng/mL, for 35 days, thentapered over 7 days and discontinued on Day 42).

In another aspect, the invention features a method of preparing arecipient, e.g., a human patient, for a graft from a donor mammal of thesame species, without the administration of whole body irradiation, andthymic irradiation. The method includes:

Administering to the recipient, one or more doses of an antibody whichdepletes T cells. The antibodies can be administered before, on orafter, the day of graft implantation. In a preferred embodiment theantibodies are administered up to or on the day of graft implantation.Suitable antibodies include, anti-CD4, anti-CD8, and anti-CD2antibodies. Anti-CD2 antibodies are preferred. For example, the methodcan include, administering to the recipient, an anti-CD2 monoclonalantibody, e.g., administering to the recipient, at least one dose, andpossibly two, three or four, doses of an anti-CD2 monoclonal antibody,e.g., before or up to the day of graft implantation. By way of example,four doses of an anti-CD2 monoclonal antibody, e.g., MEDI-507, can beadministered. The doses should be more or less evenly spaced with thelast occurring the day before transplantation, e.g., on days −4, −3, −2,−1. A dose of 0.1 mg/kg on day −4 and of 1.0 mg/kg on days −3, −2, and−1, of the anti-CD2 monoclonal antibody MEDI-507 has been found to beeffective. Analogous doses of other anti-CD2 antibodies can be used;

Optionally, administering to the recipient, a treatment which reducesthe side effects on the administration of anti-T cell antibodies, e.g.,administering an agent which suppresses the release of antibody-inducedcytokine release. By way of example, the method can includeadministering to the recipient, a steroid immunosuppressant, e.g.,methylprednisolone. The immunosuppressant can be administered prior toone or more, e.g., prior to each, or prior to the first, administrationof a T cell depleting antibody, e.g., an anti-CD2 antibody. By way ofexample, an intravenous dose of methylprednisolone sodium succinate, 8mg/kg, to a maximum dose of 500 mg has been found to be effective. Themethylprednisolone can be administered 1-4 hours (preferably, between 1and 2 hours) before antibody administration;

Optionally, administering a treatment which inhibits or depletes splenicT cells and/or B cells. Examples of such treatment include theadministration of anti-T cell antibodies, anti-B cell antibodies orimmunosuppressive agents, in sufficient dosage to inhibit or depletesplenic antibodies, or performing a splenectomy. In a preferredembodiment the method includes, performing a splenectomy on therecipient, e.g., on the day of transplantation day 0;

Administering to the recipient, donor hematopoietic progenitor cells,e.g., donor hematopoietic bone marrow cells. Mobilized donor peripheralblood progenitor cells are preferred. Hematopoietic stem cells can beadministered before, after, or on the day of transplantation (day 0). Anamount of donor hematopoietic stem cells sufficient to result in mixedchimerism in the absence of whole body irradiation and thymicirradiation is administered;

Optionally, administering to the recipient, a non-chronic course of animmunosuppressant, e.g., cyclosporine or FK506. By way of example, animmunouppressant can be administered from the day of transplant butdiscontinued after stable chimerism has been established. The dose of animmunosuppressant can be given in two phases, a first phase in which achosen level of immunosuppressant is established, e.g., a levelsufficient to inhibit graft-reactive T cells, followed by a phase inwhich the dose is tapered. E.g., cyclosporine can be administered toprovide a trough whole blood concentration of 400-500 ng/mL, for 35days, then tapered over 7 days and discontinued on Day 42); and

Administering to the recipient, an inhibitor, e.g., a blocker, of acostimulatory pathway (e.g., a blocker, e.g., an inhibitor, e.g., one orboth of a blocker of the CD40 ligand-CD40 interaction and a blocker ofthe CD28-B7 interaction). A blocker of the CD40/CD40L interaction, e.g.,an anti-CD40L antibody can be administered prior to administration of ablocker of the CD28/B7 interaction, e.g., CTLA4/Ig. The CD40/CD40Lblocker can be administered on the day donor tissue is introduced andthe CD28/B7 blocker administered 2, 3, 4 5 or more days later.

In preferred embodiments the CD40 ligand-CD40 interaction is inhibitedby administering an antibody or soluble ligand or receptor for the CD40ligand or CD40, e.g., by administering an anti-CD40L antibody, e.g., 5c8(see U.S. Pat. No. 5,474,711, hereby incorporated by reference) or anantibody with similar efficacy or an antibody whose epitope overlapsthat of 5c8. Preferably the inhibitor binds the CD40 ligand.

In preferred embodiments the CD28-B7 interaction is inhibited byadministering a soluble ligand or receptor or antibody for the CD28 orB7, e.g., a soluble CTLA4, e.g., a CTLA4 fusion protein, e.g., a CTLA4immunoglobulin fusion, e.g., CTLA4/Ig. Preferably, the inhibitor bindsB7. In preferred embodiments anti-B7-1 or anti-B7-2 antibodies areadministered.

In preferred embodiments CTLA4-Ig and an anti-CD40L antibody areadministered.

In preferred embodiments, the method includes the step of introducinginto the recipient, a graft obtained from the donor. The graft can beother than hematopoietic stem cells or bone marrow, e.g., a heart,pancreas, liver, or kidney.

In a preferred embodiment the donor is related to the recipient, e.g.,is a sibling, parent, or cousin.

In a preferred embodiment the donor is not related to the recipient.

In a preferred embodiment the graft is from a cadaver.

In a preferred embodiment the method includes:

administering a T cell depleting antibody, e.g., an anti-CD2 monoclonalantibody, e.g., administering an anti-CD2 monoclonal antibody as asingle or multiple infusions(s) prior to the graft. In one preferredembodiment, a single infusion (with or without a test dose given the daybefore) is administered a week prior to the graft. In another preferredembodiment, multiple administrations are given at regularly-spacedintervals, e.g., on days −4, −3, −2, −1;

optionally, administering a steroid immunosuppressant, e.g., prior tothe first, or each, antibody administration, e.g., administering a doseof methylprednisolone sodium succinate, prior to the first, or each,antibody administration;

optionally, performing a splenectomy on the recipient, e.g., on the dayof transplantation day 0;

administering to the recipient, donor peripheral blood progenitor cells,e.g., on the day of transplantation (day 0), in an amount sufficient toresult in mixed chimerism in the absence of whole body irradiation andthymic irradiation;

optionally, administering to the recipient, non-chronic course of animmunosuppressant, e.g., cyclosporine started on the day prior totransplantation. By way of example, starting on the day aftertransplantation (Day 1), cyclosporine can be administered to provide atrough whole blood concentration of 400-500 ng/mL, for 35 days, thentapered over 7 days and discontinued on Day 42); and

administering to the recipient, an inhibitor, e.g., a blocker, of acostimulatory pathway (e.g., a blocker, e.g., an inhibitor, e.g., one orboth a blocker of the CD40 ligand-CD40 interaction and a blocker of theCD28-B7 interaction). A blocker of the CD40/CD40L interaction, e.g., ananti-CD40L antibody can be administered prior to administration of ablocker of the CD28/B7 interaction, e.g., a soluble CTLA4, e.g., a CTLA4fusion protein, e.g., a CTLA4 immunoglobulin fusion, e.g., CTLA4/Ig. TheCD40/CD40L blocker can be administered on the day donor tissue isintroduced and the CD28/B7 blocker administered 2, 3, 4 5 or more dayslater.

In preferred embodiments the CD40 ligand-CD40 interaction is inhibitedby administering an antibody or soluble ligand for the CD40 ligand orCD40, e.g., by administering an anti-CD40 or CD40L antibody, e.g., MR-1,or an antibody which binds the MR-1 epitope. Preferably the inhibitorbinds the CD40 ligand.

In preferred embodiments the CD28-B7 interaction is inhibited byadministering a soluble ligand or antibody for the CD28 or B7, e.g.,soluble CTLA4, e.g., a soluble CTLA4 fusion protein, e.g., a CTLA4-Igfusion protein.

In preferred embodiments CTLA4-1gG or an anti-B7 antibody areadministered.

“Hematopoietic space”, as used herein, refers to a condition created inthe bone marrow which promotes engraftment of administered stem cells.The most common way of creating hematopoietic space is by irradiation ofthe bone marrow with whole body irradiation. Alternatively,hematopoietic space can be created with the use of radiomimeticchemotherapeutic agents, either alone or in combination with otherchemotherapeutic (e.g., antimetabolite and/or antineoplastic) agents. Inanother embodiment, hematopoietic space can be created with the use ofnon-radiomimetic chemotherapeutic agents. Such agents might includefludarabine or other purine or deoxynucleoside analogues, such as 2-CDA,6-MP, 6-TG, gemcitabine or cytarabine.

“Thymic space” as used herein, is a state created by a treatment thatfacilitates the migration to and/or development in the thymus of donorhematopoietic cells of a type which can delete or inactivate hostthymocytes that recognize donor antigens. It is believed that the effectis mediated by elimination of host cells in the thymus.

“Graft”, as used herein, refers to a body part, organ, tissue, or cells.Organs such as liver, kidney, heart or lung, or other body parts, suchas bone or skeletal matrix, tissue, such as skin, intestines, endocrineglands, or progenitor stem cells of various types, are all examples ofgrafts.

“Thymic irradiation”, as used herein, refers to a treatment in which atleast half, and preferably at least 75, 90, or 95% of the administeredirradiation is targeted to the thymus. Whole body irradiation, even ifthe thymus is irradiated in the process of delivering the whole bodyirradiation, is not considered thymic irradiation.

“MHC antigen”, as used herein, refers to a protein product of one ormore MHC genes; the term includes fragments or analogs of products ofMHC genes which can evoke an immune response in a recipient organism.Examples of MHC antigens include the products (and fragments or analogsthereof) of the human MHC genes, i.e., the HLA genes.

A non-chronic course of an immunosuppressant refers to theadministration of an immunosuppressant, e.g., cyclosporine or FK506. Ina preferred embodiment, the immunosuppressant is administered in anamount sufficient such that T cell dependent rejection is inhibited. Ina preferred embodiment administration dose not extend beyond 2, 4, 6,12, or 18 months after graft implantation. By way of example, startingon the day after transplantation (Day 1), the dose of cyclosporine canbe 4 mg/kg given twice a day, and adjusted to provide a trough wholeblood concentration of 400-500 ng/mL, as measured by a monoclonalantibody based assay (or the equivalent if a different assay or serumrather than whole blood are used) for 35 days, then tapered over 7 daysand discontinued on Day 42.

“Tolerance”, as used herein, refers to an inhibition of a graftrecipient's immune response which would otherwise occur, e.g., inresponse to the introduction of a nonself MHC antigen into therecipient. Tolerance can involve humoral, cellular, or both humoral andcellular responses. Tolerance, as used herein, refers not only tocomplete immunologic tolerance to an antigen, but to partial immunologictolerance, i.e., a degree of tolerance to an antigen which is greaterthan what would be seen if a method of the invention were not employed.Tolerance, as used herein, refers to a donor antigen-specific inhibitionof the immune system as opposed to the broad spectrum inhibition of theimmune system seen with immunosuppressants.

“A blocker” as used herein, refers to a molecule which binds a member ofa ligand/counter-ligand pair and inhibits the interaction between theligand and counter-ligand or which disrupts the ability of the boundmember to transduce a signal. The blocker can be an antibody (orfragment thereof) to the ligand or counter ligand, a soluble ligand(soluble fragment of the counter ligand), a soluble counter ligand(soluble fragment of the counter ligand), or other protein, peptide orother molecule which binds specifically to the counter-ligand or ligand,e.g., a protein or peptide selected by virtue of its ability to bind theligand or counter ligand in an affinity assay, e.g., a phage displaysystem.

The use of the article “a” or “an” is non limiting with regard to numberexcept where clearly indicated to be limited by the context. E.g.,methods which include administering “an” antibody or a dose can includeadministering one or more than one antibodies or doses.

Methods of the invention minimize or eliminate the need forhematopoietic space-creating treatment, e.g., irradiation, in manymethods of tolerance induction.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION Hematopoietic Stem Cells

Some methods described herein include administering a sufficiently largenumber of donor hematopoietic cells to the recipient such that, donorstem cells engraft, give rise to mixed chimerism, and induce tolerancewithout space-creating treatment, e.g., without whole body irradiationand/or thymic irradiation. The number of donor hematopoietic cellsadministered can be at least twice, at least equal to, or at least 75,50, or 25% as great as, the number of hematopoietic cells found in anadult. The number of donor hematopoietic stem cells administered to therecipient can be at least twice, at least equal to, or it least 75, 50,or 25% as great as, the number of hematopoietic stem cells found in anadult. The donor stem cells can be provided in two or more separateadministrations.

The number of donor cells administered to the recipient can be increasedby either increasing the number of stem cells provided in a particularadministration or by providing repeated administrations of donor stemcells.

Repeated stem cell administration can promote engraftment, mixedchimerism, and preferably long-term deletional tolerance in graftrecipients. Thus, the invention also includes methods in which multiplehematopoietic stem cell administrations are provided to a recipient,e.g., additional administrations other than an administration on the dayof graft transplantation. Multiple administration can substantiallyreduce or eliminate the need for hematopoietic space-creatingirradiation or chemotherapy. Administrations can be given prior to, atthe time of, or after graft implantation. In preferred embodimentsmultiple administrations of stem cells are provided prior to theimplantation of a graft. Two, three, four, five, or more administrationscan be provided. The period between administrations of hematopoieticstem cells can be varied. In preferred embodiments a subsequentadministration of hematopoietic stem cell is provided: at least twodays, one week, one month, or six months after the previousadministration of stem cells; when the recipient begins to show signs ofhost lymphocyte response to donor antigen; when the level of chimerismdecreases; when the level of chimerism falls below a predeterminedvalue; when the level of chimerism reaches or falls below a level wherestaining with a monoclonal antibody specific for a donor PBMC antigen isequal to or falls below staining with an isotype control which does notbind to PBMC's, e.g. when the donor specific monoclonal stains less than1-2% of the cells; or generally, as is needed to maintain a level ofmixed chimerism sufficient to maintain tolerance to donor antigen. Stemcells may be administered either as unmanipulated, or manipulated bonemarrow or mobilized peripheral blood stem cells. Such manipulation mightinclude the positive selection of cells demonstrating a specificphenotype (i.e. the expression of an antigen such as CD34), or thenegative selection of cells demonstrating undesirable phenotypes, suchas certain populations of T-cells. In those embodiments where subsequentadministration(s) of hematopoietic stem cells are given when therecipient begins to show signs of host lymphocyte response to donorantigen or when the level of chimerism decreases, only manipulated donorbone marrow or mobilized hematopoietic stem cells should beadministered.

One or more post graft-implantation-administrations of donor stem cellscan also be provided to minimize or eliminate the need for irradiation.Post graft administration of hematopoietic stem cell can provided: atleast two days, one week, one month, or six months after the previousadministration of stem cells; at least two days, one week, one month,six months, or at any time in the life span of the recipient after theimplantation of the graft; when the recipient begins to show signs ofrejection, e.g., as evidenced by a decline in function of the graftedorgan, by a change in the host donor specific antibody response, or by achange in the host lymphocyte response to donor antigen; when the levelof chimerism decreases; when the level of chimerism falls below apredetermined value; when the level of chimerism reaches or falls belowa level where staining with a monoclonal antibody specific for a donorPBMC antigen is equal to or falls below staining with an isotype controlwhich does not bind to PBMC's, e.g. when the donor specific monoclonalstains less than 1-2% of the cells; or generally, as is needed tomaintain tolerance or otherwise prolong the acceptance of a graft.

When multiple stem cell administrations are given one or more of theadministrations can include a number of donor hematopoietic cells whichis at least twice, is equal to, or is at least 75, 50, or 25% as greatas, the number of bone marrow cells found in an adult of the recipientspecies; include a number of donor hematopoietic stem cells which is atleast twice, is equal to, or is at least 75, 50, or 25% as great as, thenumber of bone marrow hematopoietic stem cells found in an adult of therecipient species.

Sources of Cells for Allogeneic Stem Cell Transplantation

Some methods described herein use hematopoietic stem cells, e.g.,mobilized enriched peripheral blood hematopoietic stem cells, obtaineddirectly from the donor. Other methods may require more hematopoieticcells than a donor can provide. Ex vivo expansion of hematopoietic stemcells can provide larger amounts of hematopoietic cells.

A living human donor can provide about 7.5×10⁸ bone marrow cells/kg ofrecipient. Methods of the invention can include the administration of atleast 2 or 3 times this number (per kg) and in some cases up to or atleast 10, 15, or 20 times this number. The requisite numbers of bonemarrow cells can be provided by the ex vivo expansion or amplificationof human stem cells. Ex vivo expansion is reviewed in Emerson, 1996,Blood 87:3082, hereby incorporated by reference. Methods of ex vivoexpansion are described in more detail in Petzer et al., 1996, Proc.Natl. Acad. Sci. USA 93:1470; Zundstra et al., 1994, BioTechnology12:909; and WO 95 11692 Davis et al., all of which are herebyincorporated by reference. Sources of hematopoietic stem cells includebone marrow cells, mobilized peripheral blood cells, and, whenavailable, cord blood cells. Donor peripheral hematopoietic stem cellsare preferred in some methods.

Anti-CD2 Antbodies

Methods described herein include the administration of anti-CD2monoclonal antibodies to the recipient. Anti-CD2 monoclonals suitablefor use in the methods described herein can be made by generating arodent monoclonal which is reactive with CD2 found on the surface ofhuman lymphocytes and humanizing the rodent antibody. “Human” anti-CD2made in a mouse or rat reconstituted with human immune system componentscan also be used.

Particularly preferred anti-CD2 monoclonals include those which bind anepitope which overlaps, or which is similar to, the epitope bound by ratmonoclonal anti-CD2 antibody BTI-322 or the humanized anti-CD2 antibody,MEDI-507. For example, a preferred anti-CD2 antibody is one which caninhibit the binding of BTI-322 to its epitope, inhibit the binding ofMEDI-507 to its epitope, be inhibited in binding to its epitope byBTI-322, or be inhibited in binding to its epitope by MEDI-507. BTI-322is described in U.S. 5,817,311, hereby incorporated by reference.BTI-322 has been deposited with the ATCC as accession number HB 11423.MEDI-507 is described in PCT/US97/12645 (WO9903502, published Jan. 28,1999), hereby incorporated by reference.

MEDI-507 is a humanized form of a rat IgG2b kappa monoclonal antibodyraised against human lymphocytes and reactive with the CD2 antigen onthe lymphocytes. MEDI-507 can be supplied as a solution in phosphatebuffered saline (pH 7.4) in vials containing 15 mg for intravenous use.

MEDI-507 is described below. Although this description illustrates thefunctional characteristics of MEDI-507, other anti-CD2 antibodies withthese functional properties can be used. The methods used tocharacterize MEDI-507 can applied to a candidate anti-CD2 antibody todetermine if it has similar functional properties to MEDI-507, i.e.,that it has one or more of the following properties: it binds to allhuman T cells and most NK (natural killer) cells (approximately 85% ofperipheral blood mononuclear cells); it does not react nonspecificallywith human tissues; it specifically reacts with tissue of lymphoidorigin; it inhibits in vitro T cell proliferation in response tomitogens, antigens, or allogeneic stimulator cells, preferrably atnanomolar concentrations; it can interfere with the proliferation ofactivated T cells, preferably even if added as late as three days afterinitiation of a five day mixed lymphocyte response (MLR); it does notinduce proliferation, IL-2, or interferon-γ (IFN-γ) production inlymphocytes from the majority of human peripheral blood lymphocytesamples;

it results in the production of less tumor necrosis factor-α (TNF-α)than does OKT3; it inhibits IL-2, TNF-α, and IFN-γ production inresponse to OKT3 stimulation; it causes reversible modulation of theexpression of CD2 on human peripheral blood lymphocytes in culture.

Specificity

MEDI-507 binds to all human T cells and most NK (natural killer) cells(approximately 85% of peripheral blood mononuclear cells).Immunohistochemistry was used to study the specificity of binding ofMEDI-507 with human tissues. The study concluded MEDI-507 does not reactnonspecifically with human tissues. Specific staining of tissues wasobserved only in those of lymphoid origin. This result supports theobserved specificity of this antibody for the CD2 cell surface molecule.

Effect on Target Cells: Inhibitory Properties

MEDI-507 inhibits in vitro T cell proliferation in response to mitogens,antigens, and allogeneic stimulator cells at nanomolar concentrationsand at concentrations similar to that of the rat parent antibody.MEDI-507 can interfere with the proliferation of activated T cells evenif added as late as three days after initiation of a five day mixedlymphocyte response (MLR). Interaction of MEDI-507 with T cells in vitroduring alloantigen activation results in alloantigen-specific anergy orhyporesponsiveness.

Effect on Target Cells: Activation

MEDI-507 is not an activating antibody. MEDI-507 does not induceproliferation, or IL-2 or interferon-γ (IFN-γ) production in lymphocytesfrom the majority of human peripheral blood lymphocyte samples. There isless tumor necrosis factor-α (TNF-α) produced in response to MEDI-507than to OKT3 (a murine anti-CD3 antibody used for treatment of allograftrejection). MEDI-507 inhibits IL-2, TNF-α, and IFN-γ production inresponse to OKT3 stimulation.

Effect on Target Cells: CD2 Expression

MEDI-507 causes the modulation of the expression of CD2 on humanperipheral blood lymphocytes in culture. This effect is reversible.

Pharmocokinetics and Pharmacodynamics

The pharmacokinetics and pharmacodynamics of MEDI-507 were investigatedin chimpanzees under general inhalational anesthesia. MEDI-507 wasadministered as a single daily intravenous infusion over 30 minutesgiven every other day for three doses. A dose of 0.43 mg/kg was givenexcept for the first animal who received a dose of 0.143 mg/kg for thefirst of the three doses. The highest daily dose administered wasequivalent to a human dose of ˜30 mg. There was a MEDI-507treatment-related decrease in peripheral blood lymphocyte counts. Thisoccurred following the initial dose and was sustained for three weeks,then gradually resolved, with total lymphocyte numbers returning to nearbaseline values by five weeks after dosing. Inhibition of MLR and NKactivity was observed in all groups treated with MEDI-507. The resultsof lymph node biopsies taken from these chimpanzees following the threedoses of MEDI-507 over six days of treatment revealed minimal depletionof lymphocytes in the paracortical areas of lymph nodes. Moderate firstdose effects were observed in one of two chimpanzees treated as above,and in an additional chimp receiving a single dose of 0.143 mg/kg ofMEDI-507 without steroid pre-medication. These consisted ofhypertension, muscle spasms, brief apnea, rales, and apparent headache,and resolved over a period of 24 hours. No further clinical adverseevents were observed in animals receiving additional doses withoutpre-medication. A second study of MEDI-507 in ketamine sedatedchimpanzees that were not pre-medicated did not reveal these adverseeffects, with doses of 0.6 mg/kg/day and 1.43 mg/kg/day for threeconsecutive days. Mild to moderate depletion of T cells in lymph nodeswere observed in both dose groups at 7 and 11 days after the first dose,with recovery being 42-70 days after dosing. These results supplementedearlier studies in chimpanzees using BTI-322, the rodent parent antibodyto MEDI-507. Doses of 0.143 mg/kg/day and 1.43 mg/kg/day for tenconsecutive days resulted in depletion of lymphocytes in peripheralblood and lymph nodes biopsied on the day after dosing (Day 11).Depletion was more profound at the higher dose level and predominantlyaffected the T-cell regions of the lymph node in the paracorticalregions, but left the germinal centers intact. Doses lower than thesedid not produce depletion of lymphocytes in lymph nodes, but did produceperipheral blood lymphocyte depletion. In a phase I study in newlytransplanted renal allograft recipients, the estimated steady statealpha phase t_(1/2) of MEDI-507 was 29 hours in the 0.06 mg/kg dosegroup at 49 hour in the 0.12 mg/kg dose group. Additionalpharmacokinetics have been performed in patients receiving higher dosesof MEDI-507, including a study in which MEDI-507 was administered atapproximately 0.4 mg/kg. In this study, where the beta phase t_(1/2)could be estimated, the beta elimination is approximately 6-10 days.Patients in the allograft study had received high dose steroids as partof the peri-transplant immunosuppressive regimen several hours prior tothe first dose of MEDI-507; no adverse reactions or first dose effectsafter MEDI-507 administration were observed.

In methods described herein, high dose MEDI-507 immediatelypre-transplant, as part of the conditioning regimen, will result in morecomplete T cell depletion than can be achieved with ATGAM, which hasbeen used in the monkey models. Engraftment of donor hematopoieticprogenitor cells for the induction of mixed chimerism requires a highdegree of T-cell depletion in the recipient. The dose and dosing regimenof MEDI-507 chosen for use in this protocol were based on the extent ofT-cell depletion in lymph nodes and blood in studies in chimpanzees.

Safety and Efficacy

Safety and preliminary efficacy data on the use of BTI-322, the rodentmonoclonal antibody parent to the humanized antibody MEDI-507, have beenderived from more than 130 patients in the US and Europe who have beentreated with BTI-322 at doses up to 10 mg/day for up to 20 days, forseveral indications, including prevention and treatment of solid organallograft rejection, and for the treatment of steroid resistant acuteGvHD. Aside from modest first dose reactions in some patients receivingBTI-322, which have been easily controlled and confined to the firstdose, this agent has generally been well tolerated, and no apparentincrease in infections or malignancies in treated patients, some of whomhave been followed for four years, has been noted up to the presenttime. In limited studies of renal allograft recipients receivingMEDI-507 at doses up to 0.12 mg/kg, no first dose symptoms wereobserved. One patient receiving MEDI-507 with no steroid pre-medicationfor treatment of psoriasis experienced a mild/moderate first dosereaction.

Preparation, Formulation, and Storage of MEDI-507

The antibody is produced by in vitro culture of hybridoma cells in aprotein-free medium. The crude antibody is purified by a combination ofanion exchange, cation exchange and hydrophobic interaction, and sizeexclusion chromatography. The purified bulk product is released based onthe results of in vitro potency tests, purity and identity tests, andthe absence of fungi, bacteria, mycoplasma and virus contaminants. Thefinal antibody product is vialed as a solution in phosphate bufferedsaline (pH 7.4) containing no preservatives or other excipients. Eachvial contains 15 mg of antibody in a volume of 4 mL.

Recovery and Enrichment of Peripheral Blood Hematopoietic ProgenitorCells

Methods described herein can use mobilized peripheral bloodhematopoietic stem cells. It is generally desirable to enrich suchperipheral cell preparations for CD34⁺ cells. Buoyant density separationcan be used to enrich the desired cells

The enrichment system should result in a preparation in which CD34⁺cells are enriched as compared with red blood cells, granulocytes, and Tcells.

The enrichment process provides for the recovery and enrichment ofhematopoietic progenitor cells obtained by leukapheresis of peripheralblood after cytokine mobilization. This cell population is capable ofengraftment and complete hematologic reconstitution of lethallyirradiated patients with malignancies undergoing bone marrowtransplantation. Buoyant density based enrichment can provide for theproduction of a more consistent cell product for infusion, forincreasing the viability of progenitor cells after cryopreservation, andfor permitting the infusion of hematopoietic progenitor cells with areduced risk for the development of acute graft vs. host disease in therecipient.

A cell separation kit which includes several medical grade plasticwarecomponents and a bottle of buoyant density solution (BDS), a sterilenon-pyrogenic density medium which can be formulated at differentdensities to fractionate different cell types from blood, marrow, andother somatic tissues based on their buoyant density can be used for theenrichment process. The buoyant density solution has an optimum density(1.0605 g/mL) for the isolation of hematopoietic stem cells (CD34⁺cells) from cytokine-mobilized peripheral blood.

The DENDREON™ 300 System is suitable for use with methods describedherein.

Patient Selection

Although the methods described herein are generally applicable to humanrecipients, in preferred embodiments the recipients should have one ormore, and preferably all, of the characteristics described below. Thepatient and donor profiles discussed below are provided as an exampleand are not limiting. The methods described herein can be performed onpatients (and donors) which do not fit these criteria.

Preferrably, patients should be receiving a first or second transplant,which should, but does not have to be, a living donor renal allograft.The donor-recipient pair should have HLA specificities such thatdetection of chimerism by flow cytometry using anti-HLA antibodies ispossible. Both the current and historical PRA for the current allograftshould be <20%. Patients should not have received any thymic irradiationtherapy in the past, and should not have a contraindication to radiationtherapy. The donor should be capable of undergoing cytokine-mediatedprogenitor cell mobilization and leukapheresis, as well as serving asthe kidney donor. Patients should have received one dose of a currentlyrecommended pneumococcal vaccine, and H. influenzae type b conjugatevaccine at least four weeks prior to the transplant. The donor-recipientpair should have at least one HLA antigen difference detectable by flowcytometry using anti-HLA antibodies, to permit assessment of donorchimerism.

More detailed inclusion and exclusion criteria for preferred embodimentsare presented below.

Inclusion Criteria

-   -   1. Patients (male or female) must not be less than 18 years of        age nor more than 55 years of age.    -   2. Patients must be undergoing a first or second transplant,        which must be a living donor renal allograft, and may have had        either a living donor or a cadaveric transplant as the first        transplant.    -   3. Patients with a relative contraindication to standard        transplantation, either related to prior non-compliance with the        immunosuppressive regimen, underlying disease, or other reasons,        will be preferred patients for this protocol.    -   4. Women and men must be willing to use adequate contraception,        as determined by the investigator, for the first six months        after renal transplantation.    -   5. The patient or his/her guardian and the transplant donor must        voluntarily sign informed consent forms for entry into this        study after the contents of those documents have been fully        explained to them.    -   6. The kidney donor must be capable of undergoing cytokine        mediated peripheral progenitor cell mobilization and        leukapheresis.    -   7. The recipient must have received a currently recommended        pneumococcal vaccine and Hemophilus influenzae type b-conjugate        vaccine at least 4 weeks prior to transplantation.    -   8. The patient must be capable of complying with the schedule of        study visits, especially after discontinuation of cyclosporine.

Exclusion Criteria

-   -   1. Patients receiving an ABO blood group incompatible renal        allograft.    -   2. Patients with a current or historical PRA >20%, and if a        second allograft recipient must be at least one year post-graft        loss from that initial transplant.    -   3. Patients who are leukopenic (WBC less than 2000/mm³) or        thrombocytopenic (platelet count <100,000/mm³).    -   4. Patients who are seropositive for HIV-1 or HBsAg, or        sero-negative for Hepatitis C virus with a Hepatitis C virus        sero-positive donor    -   5. Patients in heart failure or pulmonary edema.    -   6. Lactating or pregnant women. Women must have a negative serum        pregnancy test prior to study entry.    -   7. Patients with a history of cancer.    -   8. Patients with a contraindication to the long-term        administration of cyclosporine post-transplant.    -   9. Patients who have received radiation therapy in the past.    -   10. Patients with a known genetic disease or family history that        may result in greater sensitivity to the effects of irradiation,        or physical deformity that would preclude adequate shielding or        appropriate dosing during the irradiation component of the        conditioning regimen.    -   11. Patients or their donors who are, in the opinion of the        investigator, not capable of giving informed consent for the        study, either because of a language barrier or for any other        reason.

Patient Care, Evaluations, and Surgical Methods

A complete physical examination of both the donor and recipient shouldbe performed. The results of the physical exam performed on the donorduring the pre-transplant evaluation process may be used in lieu of anew exam, if there has been no notable change in the donor's physicalcondition. Abnormalities noted on physical examination of the patientand donor should be recorded.

The surgical techniques related to the renal transplant and (optional)splenectomy can be accomplished according to the surgeon's clinicaljudgement and experience using standard techniques. Procedures performedon the donor, including cytokine mobilization of peripheral bloodprogenitor cells and leukapheresis are described herein, but the donornephrectomy should be accomplished according to the surgeon's clinicaljudgement and experience.

Recipients should be evaluated during the first three weekspost-transplant for the acute safety and tolerability of theconditioning regimen, including evaluation for neutropenia,thrombocytopenia, lymphopenia and nausea as well as acute side effectsthat may be associated with anti-CD2 antibodies, e.g., MEDI-507. Theoccurrence of other opportunistic and invasive bacterial infections andmalignancies should be assessed at 3 and 6 months, and, 1 and 2 yearspost-transplant. Allograft rejection, renal function, graft vs. hostdisease, and graft and patient survival, will be assessed at 1, 3, 6,12, and 24 months post-transplant. The allograft donor will undergohematologic assessment following the progenitor cell mobilizationprocedures at 1, 3, 6, and 12 months post-donation, an assessment of thetolerability and safety of the G-CSF treatment, and renal function.

Evaluation of donor cell chimerism by flow cytometry should be donepre-transplant, and at 1, 3, 7, 10, 14, 21, 28, 42, and 56 dayspost-transplant in transplant recipients. If chimerism is still presentat 56 days, patients should be evaluated every 30 days thereafter forpersistence of chimerism, until no longer detectable, or until one yearpost-transplant. Peripheral blood lymphocyte subsets should be evaluatedat the same time points. A lymph node biopsy should be taken from therenal bed at the time of transplantation, and assessed by routinehistology, by immunohistochemistry, and by flow cytometry for lymphocytesubsets. Mixed lymphocyte reaction (MLR) should be performed atbaseline, and every 3 months for one year post-transplant, usingirradiated frozen donor lymphocytes as stimulator cells, and usingstored pooled third party lymphocytes as control stimulator cells,respectively, with recipient lymphocytes as responders.

Kits

Kits for use with methods described herein are provided. A kit willinclude the supplies necessary for the conditioning of one patient,including materials for buoyant density separation of hematopoietic stemcells, e.g., a DENDREON™ 300 System, and a supply of anti-CD2 humanizedmonoclonal antibody, e.g., MEDI-507 monoclonal antibody. The twocomponents of the kit require different storage conditions, thematerials for cell separation at room temperature (20-25° C.), andmonoclonal antibody at 2-8° C. Kits will be assigned to specificpatients. No component of any kit should be exchanged between kits, andkits may not be exchanged between patients.

Example 1 Allogeneic Transplantation With Whole Body Irradiation andThymic Irradiation

Methods described include a series of procedures and treatmentsincluding total body and thymic irradiation, antibody administration,hematopoietic progenitor cell infusion, and splenectomy.

The renal allograft recipient will receive:

300 cGy (150 cGy on each of two successive days) of whole bodyirradiation;

700 cGy of thymic irradiation;

one or more doses (preferably, four doses) of an anti-CD2 monoclonalantibody, e.g., MEDI-507, on days −4, −3, −2, −1, at a dose of 0.1 mg/kgon day −4 and of 1.0 mg/kg on days −3, −2, and −1, (this can be precededby 1-4 hours by a single intravenouse dose of methylprednisolone sodiumsuccinate, 8 mg/kg, to a maximum dose of 500 mg);

a splenectomy on the day of transplantation day 0;

donor peripheral blood progenitor cells will be infused on the day oftransplantation day 0; and

a 42-day course of cyclosporine started on the day prior totransplantation.

The regimen begins six days prior to the day of transplantation, whichis designated Day 0. Days pre-transplant are designated by negativenumbers in descending order, while days post-transplant are designatedby positive numbers in ascending order. The method is described in moredetail below.

Cytokine Mobilization of Hematopoietic Progenitor cells andLeukapheresis of the Donor

Renal donors will undergo treatment with recombinant human G-CSF(filgrastim), 6-10 micrograms/kg/day subcutaneously for 5-7 days tomobilize hematopoietic progenitor cells into the peripheral blood wherethey will be collected by a 3-4 blood volume (15-18L) leukapheresisusing the standard techniques. Cytokine mobilization should bemonitored, and the day of leukapheresis will be determined, by the CD34+cell count and total white blood cell count in peripheral blood. Theleukapheresis product should then be further processed by separatinghematopoietic cells by buoyant density, e.g., by using a system such asthe DENDREON™ 300 System, then cryopreserved until needed for infusioninto the recipient at the time of transplantation. In one embodiment,cells may be cryopreserved with 4% human serum albumin using 10%dimethylsulfoxide (DMSO) with controlled rate freezing. In anotherembodiment, an appropriate amount of donor plasma may be used in lieu ofhuman serum albumin. A minimum of 2×10⁸ cells/kg, containing at least5×10⁶ CD34⁺ cells/kg after processing are required, and if necessary mayrequire more than one leukapheresis procedure to be performed. Theprocedure used is similar to that used for autologous or allogeneicprogenitor cell transplantation. Cytokine treatment and leukapheresisshould be completed no less than two weeks prior to the transplant toprovide adequate time for hematologic recovery and return to baselinestatus prior to renal donation. The total WBC count of the donor shouldnot exceed 70×10³/mm³. If this level is reached, the dose of filgrastimshould be reduced and the WBC count followed daily until it falls belowthis value. Every effort should be made to collect an adequate cellproduct from these patients, however, unless clinically contraindicated.

Cytokine Mobilization of Hematopoietic Progenitor cells andLeukapheresis of the Recipient

The same procedure as outlined for the donor will be performed on therecipient at least 2 weeks prior to transplantation to store frozenprogenitor cells that will be available in case of slow recovery ofhematopoiesis following administration of the conditioning regimen. Inthis case, 2×10⁶ CD34+ cells/kg will be stored. Buoyant densitypurification is not required for this cell product. The decision to usethese cells for reconstitution of the recipient will be based on theclinical judgement of the investigator and his local hematologyconsultant, in consultation with the study medical monitor.

Buoyant Density Based Enrichment of Progenitor Cells

Hematopoietic cells should be enriched by a buoyant density-basedmethod. Colloidal silica solution (1.0605 g/mL) (BDS 60) formulated forthe recovery, at the density interface, of CD 34⁺ cells from peripheralblood should be used. This material has not been formulated for therecovery of CD 34⁺ cells from bone marrow, and thus, can only be usedfor recovery of progenitor cells from blood after cytokine mobilization.A kit, such as the DENDREON™ 300 System, which includes sterile,centrifugation and washing containers and tubing that allow processingof cells in a closed manner, can be used in the enrichment process. Aplastic insert in the separation container facilitates the trapping ofunwanted cells below the density interface where the CD 34⁺ cells arerecovered. All cell processing must be done using sterile techniqueunder aseptic conditions in a properly equipped laboratory withexperience in the preparation of hematopoietic progenitor cells forhuman progenitor cell and bone marrow transplantation. The procedureoutlined below is to be followed for the preparation of donor cells forinfusion. The cells are separated at about 850 g. Sample treatment is asfollows:

Sample Preparation

-   -   Use the mobilized apheresis product. If anticoagulant is        necessary, an anticoagulant such as Anticoagulant Citrate        Dextose USP formula A (ACD-A) may be used. Determine the number        of nucleated cells and hematocrit on an aliquot of the product.        Centrifuge the product to remove plasma and resuspend the pellet        in 250 mL of 0.9% saline (Ca, Mg free). The maximum number of        nucleated cells per device is 5×10¹⁰ cells, with a hematocrit of        no greater than 15%.

Device Preparation

-   -   (The following instructions are provided for use with the        DENDREON™ 300 System or similar devices. Other devices can be        used as well, though the exact protocol may differ.)    -   Shake the bottle of BDS60 solution to mix the contents well.        Using tubing set A, connect the separation device to the bottle        of BDS60.    -   Use tubing set to connect the apheresis bag to the device.    -   Open the separation container air vent.    -   Fill the lower chamber just to the top of the insert with BDS60        colloidal silica solution. Tilt the device by about 1 cm with        the vent side up and fill the upper chamber with 250 mL of the        cell suspension. Do not fill the chamber to above the fill line        on the side. Finish filling the chamber with the remaining        colloidal silica solution by underlaying below the cell        suspension through port A, and close the air vent after removing        all the tubing.    -   Disconnect the tubing sets, and centrifuge the chamber at 850 g        for 30 minutes at ambient temperature (18-22° C.). Do not use        the brake on the centrifuge.

Harvest the Cells

-   -   (The following instructions are provided for use with the        DENDREON™ 300 System or similar devices. Other devices can be        used as well, though the exact protocol may differ.)    -   Open the Separation Container air vent upon removal from the        centrifuge. Connect a tubing set between red port B on the        separation chamber and white port C on the Wash Container.    -   Invert the separation container. Open the clamp of the tubing        and the air vent of the wash container and fill the wash        container. Do not exceed the fill line on the side of the        chamber. Disconnect the tubing and close the air vent on the        Separation Container and the Wash Container.

Wash the Cells

-   -   (The following instructions are provided for use with the        DENDREON™ 300 System or similar devices. Other devices can be        used as well, though the exact protocol may differ.)    -   Wash the cells using a conical centrifuge bucket adapter.    -   Centrifuge the wash container at 850 g for 15 minutes at ambient        temperature. Using a new Tubing Set connected to white port C,        and opening the air vent, invert and drain the supernatant.    -   Using a new Tubing Set connect a bag of 0.9% saline (Ca, Mg        free) to the Wash Container, and fill to the fill line.        Disconnect the tubing, close the port, and air vent, and        resuspend the pellet by inversion and using a vortex mixer.    -   Immediately centrifuge the cell suspension at 550 g for 10        minutes at ambient temperature. Do not use the brake.    -   Attach Tubing set C and drain the supernatant. Remove and        discard the tubing.

Pellet Recovery

-   -   (The following instructions are provided for use with the        DENDREON™ 300 System or similar devices. Other devices can be        used as well, though the exact protocol may differ.)    -   Use a 60 cc syringe to deliver a specified volume of culture        medium to the Wash Container.    -   Close the port and swirl the Wash Container to resuspend the        cells.    -   Attach Tubing Set B to the Container and drain the cell        suspension into a storage bag.

Cryopreservation of the CD34⁺ Cell Preparation

The CD34+ enriched cell product should be cryopreserved using 10% DMSOwith 4% human serum albumin and controlled rate freezing. Cells are thentransferred to the vapor phase of a liquid N₂ freezer until used. Threesmaller aliquots of cells should also be cryopreserved for subsequentCFU assays.

Characterization of the Leukapheresis Product

The leukapheresis product should be characterized as to the totalvolume, hematocrit, total nucleated cell content, total CD3+ T-cellcontent, and total CD34+ cell content, as well as CFU-GM content, at thetime of harvest, prior to enrichment by buoyant density. These sametests should be performed on the cell product harvested from the densityinterface prior to cryopreservation. If more than one leukapheresis isperformed, these tests should be done on each harvest. In addition,aliquots of cells from the original and post-buoyant density processedmaterial should be tested for sterility, and the results of such testsobtained prior to infusion of cells into the recipient.

Whole Body Irradiation (Days −6 and −5)

The 300 cGy dose of whole body irradiation (WBI) is administered in 2fractions of 150 cGy each, on 2 successive days (Days −6 and −5).Patients will be premedicated, e.g., with odansetron hydrochloride(Zofran) 32 mg W over 30 min. prior to each days irradiation, and asneeded after irradiation to treat nausea. Patients will be irradiatedusing an AP/PA approach while lying supine on the table. Two fields willbe used, with matching at the umbilicus. The upper field will have itsupper border just below the orbit, and the lower field will have itslower border at the knees. The dose will be calculated at the midplaneat the umbilicus. A 6 MV machine is optimal, but energies of 4-10 MV canbe used, the latter with beam spoiling. The dose rate used will be themaximum permitted by the machine, unless a dedicated WBI machine isavailable to administer a dose rate of 10cGy per minute, in which casethat machine should be used. In men the testes will be shielded. Womenwill be offered the option for laparoscopic clipping and fixing of oneovary to permit shielding. No lung shielding will be used. The detailsregarding the field sizes, dose calculation, energy, beam spoiling, anddose rate will be recorded on the CRF.

Thymic Irradiation (Day −1)

700 cGy of thymic irradiation can be administered in a single dose onDay −1. A field size of approximately 8 cm wide and 10 cm inlongitudinal dimension should be used, with the midpoint of the upperedge of the field at the sternal notch. The dose should be calculated ata depth of approximately 6 cm, guided by results of a lateral chestroentgenogram for the approximate location of the thymus. A 10 MVmachine at maximum dose rate should be used. No premedication for nauseashould be required. The details regarding the field sizes, dosecalculation, energy, beam spoiling, and dose rate will be recorded onthe CRF.

Administration of Anti-CD2 Monoclonal Antibody (Days −4, −3, −2, and −1)

The first dose of anti-CD2 monoclonal antibody, e.g., MEDI-507, whichmay be administered on Day −4, or prior to that, e.g., Day −7, should be0.1 mg/kg, administered over 30 minutes, and preceeded, by 1-4 hours, bythe administration of intravenous methylprednisolone sodium succinate, 8mg/kg up to a maximum dose of 500 mg, along with diphenhydramine 50 mgp.o., and acetominophen, 650 mg, p.o. The second (and subsequent, ifany) doses (Days −3, −2 and −1) should be 1 mg/kg, administered over 2hours, without steroid pre-medication. Diphenhydramine and acetominophenmay be administered prior to the second and subsequent doses, however.

The prepared 0.1 mg/kg and 1 mg/kg doses of monoclonal antibody shouldbe supplied from the pharmacy to the person who will administer thedrug. For the first dose (0.1 mg/kg), monoclonal antibody should beadministered over 30 minutes using an infusion pump. The solution shouldbe prepared to a final concentration of 0.1-0.3 mg/mL (30-100 mL).Subsequent doses are 1 mg/kg. These doses should be administered over 2hours at a permissible concentration of 0.3-1.8 mg/mL (50-100 mLinfusion volume), using an infusion pump.

The actual start and stop times of antibody administration, should berecorded on the appropriate case report form. Drug doses should beadministered at the same time each day, so that there are 24 hoursbetween doses. Antibody should be administered several hours afterdialysis, if possible.

Since compatibility with other intravenously administered medications isnot known, the monoclonal antibody solutions should not be infusedthrough a common intravenous line used for other medications, unless theline is flushed prior to and after administration of the monoclonalantibody.

MEDI-507 Preparation

(The following instructions are provided for MEDI-507. Other anti-CD2antibodies can be used, though the exact protocol may differ.)

-   -   1. Bring an appropriate number of vials for each dose to room        temperature (approximately 30 minutes) The number of vials        supplied in each patient-specific kit are appropriate for the        patient's weight. One vial is needed for the 0.1 mg/kg dose on        the first dosing day (Day −7 or Day −4).

TABLE 0-1 Number of vials needed for 1 mg/kg doses. Patient Weight #Vials per dose Total Dose <40 kg 2 30 mg 41-50 kg 3 45 mg 51-60 kg 4 60mg 61-80 kg 5 75 mg 81-100 kg 6 90 mg

-   -   2. Inspect each vial visually for precipitate and discoloration.        If there is precipitate and/or discoloration, do not use the        vial.    -   3. Preparation of MEDI-507 for Injection        -   One vial (15 mg in 4 mL) is required for the first dose of            MEDI-507 (0.1 mg/kg). Once the appropriate volume is            removed, the remaining MEDI-507 must be discarded, after            appropriate drug accountability information is completed.            The appropriate volume of MEDI-507 should be removed using a            10 mL syringe through a sterile low protein binding filter            (0.2 micrometers pore size) using aseptic technique. Discard            the filter. Dilute the antibody solution to a final volume            of 30-100 mL in a syringe pump for infusion. Steroid            premedication (see above, and Section 10) is required prior            to administration of the first dose, which is infused over            30 minutes. For subsequent doses draw the required total            volume of antibody solution into a 60 mL syringe through a            sterile low protein binding filter (0.2 micrometer pore            size) using aseptic technique. Discard the filter. Dilute            the antibody solution to a final volume of 50-100 mL in the            IV infusion bag or syringe pump. The 50-100 mL volume of the            MEDI-507 solution is infused over 2 hours. Pre-medication            with 650 mg of acetominophen and 50 mg of diphenhydramine            may precede administration of MEDI-507 by two hours. This            procedure should be followed for each of the subsequent            MEDI-507 infusions.

Donor Progenitor Cell Infusion, Splenectomy and Renal Transplantation(Day 0)

Donor progenitor cells are thawed and prepared for infusion according tostandard procedures. The performance of a splenectomy is optional priorto the renal transplant and progenitor cell infusion, according tostandard surgical techniques. If the splenectomy is performed, thespleen should be sent for routine histopathology, and bothimmunofluorescence and flow cytometry evaluations. The renal transplantis performed according to standard surgical techniques, preferably usingan iliac fossa, extraperitoneal approach and utilizing aureteropyelostomy with the recipient ureter if surgically feasible.

Administration of an immunosupressant, e.g., Cyclosporine, and Use ofCorticosteroids

Cyclosporine, either Neoral® or Sandimmune® or equivalent should beadministered orally starting on Day −1. The initial dose should be 6mg/kg given twice, approximately 12 hours apart, followed by a singledose of 4 mg/kg given late in the evening on the day of transplantation(Day 0). Starting on the day after transplantation (Day 1), the doseshould be 4 mg/kg given twice a day, and adjusted to provide a troughwhole blood concentration of 400-500 ng/mL, as measured by a monoclonalantibody based assay, or the equivalent if a different assay or serumrather than whole blood are used, to determine trough cyclosporineconcentrations. Cyclosporine should be continued for 35 days, thentapered over 7 days and discontinued on Day 42, if criteria forcyclosporine are not met.

In order to further protect the patient from possible allograftrejection post-transplant, oral prednisone (or an equipotent dose ofmethyl prednisone) should be administered starting on Day 22, if thepatient has not exhibited 1% lymphocytic chimerism at any time duringthe preceeding 21 days post-transplant. The dose of prednisone will be0.5 mg/kg once daily from Days 22-42, then tapered to 0.1 mg/kg. Becausecorticosteroids may interfere with the induction of tolerance, steroidsshould only be administered under circumstances outlined here. Patientsadministered steroids would not be eligible for cyclosporine withdrawal,and so will continue on this regimen indefinitely.

Discontinuation of the Immunosupressant

Patients will be considered for the discontinuation of cyclosporine atDay 42 post-transplant if they meet the following criteria:

1. No clinically significant acute allograft rejection prior to thisdate.2. The prior demonstration of donor lymphocytic chimerism at a level of1% or greater in peripheral blood.3. Adequate renal function as assessed by serum creatinine <2.5 mg/dl atday 35, and not varying by more than 20% for 1 month preceding Day 42,unless clearly related to a known cause of renal dysfunction such ascyclosporine toxicity, a urologic complication, other drug toxicity,etc.

Patients will be followed intensively for evidence of allograftrejection following discontinuation of cyclosporine. Serum creatininewill be measured every other day for the first two weeks afterdiscontinuation, then twice a week for one month, then once a week forone month. Measurements will then be done once every two weeks for theremainder of the first year, then according to the routinepost-transplant care prescribed to all allograft recipients

Clinical Laboratory Tests

Baseline clinical laboratory tests should be done within 24 hours priorto initiation of the conditioning regimen (Day −6) for the recipient.Two baseline hematologic evaluations will be done on successive days(these follow by about two weeks the completion of the mobilization andleukapheresis of the recipient). The hematology, tests should include:RBCS, WBCs with differential, hemoglobin, hematocrit, and plateletcount. Blood chemistry should include: ALT, AST, BUN, serum creatinine,blood glucose, bilirubin, albumin, total protein, and potassium; andtests for anti-CD2 monoclonal antibody, e.g., MEDI-507 antibodies.

Baseline Immunologic Assessment of the Recipient

Lymphocyte subsets should be evaluated on 2 successive days, as for thehematologic evaluation, at least two weeks after completion of cytokinemobilization and leukapheresis. Mixed Lymphocyte Reaction (MLR) withdonor and 3rd party stimulator cells should be performed.

Evaluation of the Transplant Donor

Baseline clinical laboratory tests must be done prior to the infusion ofthe first dose of filgrastim for mobilization of progenitor cells. Thedonor will, in addition, have daily CBCs and CD34⁺ cell counts duringcytokine mobilization. A further hematologic assessment of the donorshould be performed on the day prior to start of conditioning of therecipient to assure that donation will be possible on the designatedday. Tests should include: Hematology: RBC's, hemoglobin, hematocrit,WBCs with differential and platelet count; and Blood Chemistry: ALT,AST, BUN, creatinine, blood glucose, bilirubin, albumin, total proteinand potassium.

Creating Hematopoietic Space by Administering to a Recipient One or MoreChemotherapeutic Agents

Hematopoietic space can be created, in the absence of irradiation, byadministering to a recipient one or more chemotherapeutic agents, e.g.,cyclophosphamide, busulfan, fludararabine, or any combination thereof.Preferably, one or more of the chemotherapeutic agents has aradiomimetic mechanism of action (i.e. interacts with DNA in a mechanismsimilar to ionizing radiation). In one embodiment, a single-agentchemotherapeutic regimen is administered. For example, cyclophosphamidecan be administered (preferably, intravenously) at a dose of 50mg/kg/day for three consecutive days (e.g., cyclophosphamide can beadministered on days −5, −4 and −3 prior to administration of stem cellsor bone marrow on day 0). Alternatively, a combination of agents, e.g.,cyclophosphamide, busulfan and/or fludararabine, can be used. Thecombination of agents can be administered sequentially or concurrently.Preferably, the combination is administered concurrently. Preferably,the cyclophosphamide is administered intravenously.

Example 2 Allogeneic Transplantation With Thymic Irradiation and HighDose Donor Hematopoietic Stem Cell Administration

Methods described include a series of procedures and treatmentsincluding thymic irradiation, antibody administration, high dosehematopoietic progenitor cell infusion, and splenectomy. Unlessotherwise specified the methods are the same or analogous to those usedin Example 1.

-   -   The renal allograft recipients (which do not receive whole body        irradiation) receive:

approximately 1,000 cGy of thymic irradiation;

single or repeated (e.g., four) doses of an anti-CD2 monoclonalantibody, e.g., MEDI-507, prior to transplantation, e.g., on days −4,−3, −2, −1, at, e.g., a dose of 0.1 mg/kg on day −4 and of 1.0 mg/kg ondays −3, −2, and −1, (optionally to be preceded by 1-4 hours by a singleintravenouse dose of methylprednisolone sodium succinate, 8 mg/kg, to amaximum dose of 500 mg);

an optional splenectomy on day 0;

the administration of a high dose of donor peripheral blood progenitorcells on day 0;

optionally, a 42-day course of cyclosporine started on the day prior totransplantation.

The regimen begins six days prior to the day of transplantation, whichis designated Day 0. Days pre-transplant are designated by negativenumbers in descending order, while days post-transplant are designatedby positive numbers in ascending order. The method is described in moredetail below.

Cytokine Mobilization of Hematopoietic Progenitor cells andLeukapheresis of the Donor

Hematopoietic Progenitor Cells for Use in this Step can be Produced byIn Vitro Expansion of Mobilized Peripheral Progenitor Cells.

Cytokine Mobilization of Hematopoietic Progenitor cells andLeukapheresis of the Recipient

At least 2 weeks prior to transplantation, recipient hematopoietic stemcells are mobilized to store frozen progenitor cells that will beavailable in case of slow recovery of hematopoiesis followingadministration of the conditioning regimen. This can be performedessentially as described in Example 1 above.

Buoyant Density Based Enrichment of Progenitor Cells

The step is similar to the analogous step in Example 1.

Cryopreservation of the CD34⁺ Cell Preparation

The CD34+ enriched cell product may be cryopreserved with 4% human serumalbumin (or an appropriate amount of donor plasma) using 10%dimethylsulfoxide (DMSO) with controlled rate freezing. Cells are thentransferred to the vapor phase of a liquid N₂ freezer until used. Threesmaller aliquots of cells should also be cryopreserved for subsequentCFU assays.

Characterization of the Stem Cell Preparation

The stem cell preparation should be characterized as to the totalvolume, hematocrit, total nucleated cell content, total CD3+T-cellcontent, and total CD34+ cell content, as well as CFU-GM content, at thetime of harvest, prior to enrichment by buoyant density.

Whole Body Irradiation

The use of a higher level of thymic irradiation and a higher level ofdonor hematopoietic stem cells eliminates the need for whole bodyirradiation.

Thymic Irradiation (Day −1)

1,000 cGy of thymic irradiation can be administered in a single dose onDay −1. A field size of approximately 8 cm wide and 10 cm inlongitudinal dimension should be used, with the midpoint of the upperedge of the field at the sternal notch. The dose should be calculated ata depth of approximately 6 cm, guided by results of a lateral chestroentgenogram for the approximate location of the thymus. A 10 MVmachine at maximum dose rate should be used. No premedication for nauseashould be required. The details regarding the field sizes, dosecalculation, energy, beam spoiling, and dose rate will be recorded onthe CRF.

Administration of Anti-CD2 Monoclonal Antibody (Days −4, −3, −2, and −1)

The first dose of anti-CD2 monoclonal antibody, e.g., MEDI-507, (Day −4)should be 0.1 mg/kg, administered over 30 minutes, and preceeded, by 1-4hours, by the administration of intravenous methylprednisolone sodiumsuccinate, 8 mg/kg up to a maximum dose of 500 mg, along withdiphenhydramine 50 mg p.o., and acetominophen, 650 mg, p.o. The secondand subsequent doses (Days −3, −2 and −1) should be 1 mg/kg,administered over 2 hours, without steroid pre-medication.Diphenhydramine and acetominophen may be administered prior to thesecond and subsequent doses, however.

In general, the administration is similar to that described above inExample 1. Optionally, one or more additional doses of anti-CD2 antibodycan be administered.

High Dose Donor Progenitor Cell Infusion, Splenectomy and RenalTransplantation (Day 0)

Donor progenitor cells are thawed and prepared for infusion according tostandard procedures. An optional splenectomy may be performed prior tothe renal transplant and progenitor cell infusion, according to standardsurgical techniques. If the spleen is removed, it is spleen is sent forroutine histopathology, and both immunofluorescence and flow cytometryevaluations. The renal transplant is performed according to standardsurgical techniques, preferably using an iliac fossa, extraperitonealapproach and utilizing a ureteropyelostomy with the recipient ureter ifsurgically feasible.

Administration of an immunosupressant, e.g., Cyclosporine, and Use ofCorticosteroids

This step is optional. An immunosupressant, e.g., cyclosporine, isadministered essentially as described in Example 1.

Discontinuation of the Immunosupressant

Discontinuation criteria are similar to those discussed in Example 1.

Clinical laboratory tests, baseline immunologic assessment of therecipient, evaluation of the transplant donor are performed essentiallyas is described in Example 1.

Example 3 Allogeneic Transplantation With High Dose Donor HematopoieticStem Cell Administration and Blockade of Costimulation

Methods described include a series of procedures and treatmentsincluding antibody administration, high dose hematopoietic progenitorcell infusion, blockade of costimulation, and splenectomy. Unlessotherwise specified the methods are the same or analogous to those usedin Examples 1 and 2.

Donor renal allograft recipients (which do not receive whole body orthymic irradiation) receive:

repeated (e.g., four) doses of an anti-CD2 monoclonal antibody, e.g.,MEDI-507, prior to transplantation, e.g., on days −4, −3, −2, −1, at,e.g., a dose of 0.1 mg/kg on day −4 and of 1.0 mg/kg on days −3, −2, and±1, (optionally to be preceded by 1-4 hours by a single intravenousedose of methylprednisolone sodium succinate, 8 mg/kg, to a maximum doseof 500 mg;

a splenectomy on day 0;

the administration of a high dose of donor peripheral blood progenitorcells;

administration of an agent which blocks costimulation; optionally, a42-day course of cyclosporine on the day prior to transplantation.

The regimen begins six days prior to the day of transplantation, whichis designated Day 0. Days pre-transplant are designated by negativenumbers in descending order, while days post-transplant are designatedby positive numbers in ascending order. The method is described in moredetail below.

Cytokine Mobilization of Hematopoietic Progenitor cells andLeukapheresis of the Donor

Hematopoietic Progenitor Cells for Use in this Step can be Produced byIn Vitro Expansion of Mobilized Peripheral Progenitor Cells.

Cytokine Mobilization of Hematopoietic Progenitor cells andLeukapheresis of the Recipient

At least 2 weeks prior to transplantation, recipient hematopoietic stemcells are mobilized to store frozen progenitor cells that will beavailable in case of slow recovery of hematopoiesis followingadministration of the conditioning regimen. This can be performedessentially as described in Example 1 above

Buoyant Density Based Enrichment of Progenitor Cells

The step is similar to the analogous step in Example 1.

Cryopreservation of the CD34⁺ Cell Preparation

The CD34+ enriched cell product may be cryopreserved with 4% human serumalbumin (or an appropriate amount of donor plasma) using 10%dimethylsulfoxide (DMSO) with controlled rate freezing. Cells are thentransferred to the vapor phase of a liquid N₂ freezer until used. Threesmaller aliquots of cells should also be cryopreserved for subsequentCFU assays.

Characterization of the Stem Cell Preparation

The stem cell preparation should be characterized as to the totalvolume, hematocrit, total nucleated cell content, total CD3+T-cellcontent, and total CD34+ cell content, as well as CFU-GM content, at thetime of harvest, prior to enrichment by buoyant density.

Whole Body Irradiation

The use of a higher level of donor hematopoietic stem cells andcostimulatory blockade allow whole body irradiation to be omitted fromthe method.

Thymic Irradiation (Day −1)

The use of a higher level of donor hematopoietic stem cells andcostimulatory blockade allow thymic irradiation to be omitted from themethod.

Administration of Anti-CD2 Monoclonal Antibody (Days −4, −3, −2, and −1)

The first dose of anti-CD2 monoclonal antibody, e.g., MEDI-507, (Day −4)should be 0.1 mg/kg, administered over 30 minutes, and preceeded, by 1-4hours, by the administration of intravenous methylprednisolone sodiumsuccinate, 8 mg/kg up to a maximum dose of 500 mg, along withdiphenhydramine 50 mg p.o., and acetominophen, 650 mg, p.o. The secondand subsequent doses (Days −3, −2 and −1) should be 1 mg/kg,administered over 2 hours, without steroid pre-medication.Diphenhydramine and acetominophen may be administered prior to thesecond and subsequent doses, however.

In general, the administration is similar to that described above inExample 1. Optionally, one or more additional doses of anti-CD2 antibodycan be administered.

Donor Progenitor Cell Infusion, Splenectomy and Renal Transplantation(Day 0)

Donor progenitor cells are thawed and prepared for infusion according tostandard procedures. An optional splenectomy may be performed prior tothe renal transplant and progenitor cell infusion, according to standardsurgical techniques. If removed, the spleen can be sent for routinehistopathology, and both immunofluorescence and flow cytometryevaluations. The renal transplant is performed according to standardsurgical techniques, preferably using an iliac fossa, extraperitonealapproach and utilizing a ureteropyelostomy with the recipient ureter ifsurgically feasible.

Administration of an Agent that Blocks Costimulation

The recipient can be administered an agent which blocks a costimulatorypathway, e.g., one or both of an agent which inhibits the CD40ligand-CD40 interaction and an agent which inhibits the CD28-B7interaction. Such agents can include monoclonal antibodies directedagainst one of the lignads, or a soluble form of one of the ligands,e.g., anti-CD40 or anti-CD40 ligand antibody, or CTLA4-1gG.

Administration of an immunosupressant, e.g., Cyclosporine, and Use ofCorticosteroids

This step is optional An immunosupressant, e.g., cyclosporine, isadministered essentially as described in Example 1.

Discontinuation of the Immunosupressant

Discontinuation criteria are similar to those discussed in Example 1.

Clinical laboratory tests, baseline immunologic assessment of therecipient, evaluation of the transplant donor are performed essentiallyas is described in Example 1.

Other Embodiments

The methods described herein for inducing tolerance to, or promoting theacceptance of, an allogeneic antigen or allogeneic graft can be usedwhere, as between the donor and recipient, there is any degree ofmismatch at MHC loci or other loci which influence graft rejection.Preferably, there is a mismatch at least one MHC locus or at least oneother locus that mediates recognition and rejection, e.g., a minorantigen locus. With respect to class I and class II MHC loci, the donorand recipient can be: matched at class I and mismatched at class II;mismatched at class I and matched at class II; mismatched at class I andmismatched at class II; matched at class I, matched at class II. In anyof these combinations other loci which control recognition andrejection, e.g., minor antigen loci, can be matched or mismatched. Asstated above, it is preferable that there is mismatch at least onelocus. Mismatched at MHC class I means mismatched for one or more MHCclass I loci, e.g., in the case of humans, mismatched at one or more ofHLA-A, HLA-B, or HLA-C. Mismatched at MHC class II means mismatched atone or more MHC class II loci, e.g., in the case of humans, mismatchedat one or more of a DP α, a DPβ, a DQ α, a DQ β, a DR α, or a DR.

The methods described herein for inducing tolerance to an allogeneicantigen or allogeneic graft can be used where, as between the donor andrecipient, there is any degree of reactivity in a mixed lymphocyteassay, e.g., wherein there is no, low, intermediate, or high mixedlymphocyte reactivity between the donor and the recipient. In preferredembodiments mixed lymphocyte reactivity is used to define mismatch forclass II, and the invention includes methods for performing allogeneicgrafts between individuals with any degree of mismatch at class II asdefined by a mixed lymphocyte assay. Serological tests can be used todetermine mismatch at class I or II loci and the invention includesmethods for performing allogeneic grafts between individuals with anydegree of mismatch at class I and or II as measured with serologicalmethods. In a preferred embodiment, the invention features methods forperforming allogeneic grafts between individuals which, as determined byserological and or mixed lymphocyte reactivity assay, are mismatched atboth class I and class II.

The methods of the invention are particularly useful for replacing atissue or organ afflicted with a neoplastic disorder, particularly adisorder which is resistant to normal modes of therapy, e.g.,chemotherapy or radiation therapy. The methods of the invention are alsoparticularly useful in replacing tissue or organ with a patient who isbelieved to be, based on past experience or current expectations,unreliable with respect to the careful self-administration of chronicimmunosuppressive regimens which would otherwise be required followingthe transplantation of a mismatched mammalian organ or tissue. Methodsof the invention can be used for inducing tolerance to a graft, e.g., anallograft, e.g., an allograft from a donor which is mismatched at one ormore class I loci, at one or more class II loci, or at one or more lociat each of class I and class II. In preferred embodiments: the graftincludes tissue from the digestive tract or gut, e.g., tissue from thestomach, or bowel tissue, e.g., small intestine, large intestine, orcolon; the graft replaces a portion of the recipient's digestive systeme.g., all or part of any of the digestive tract or gut, e.g., thestomach, bowel, e.g., small intestine, large intestine, or colon.

In any of the methods described herein, particularly primate or clinicalmethods, it is preferable to form mixed chimerism as opposed to entirelyreplacing the recipient's stem cells with donor cells.

Blockers of the CD40 ligand-CD40 or CD28-B7 interactions (or both) canbe administered repeatedly. E.g., blockers can be administered one, two,three, or more times prior to donor bone marrow transplantation.Typically, a pre-bone marrow transplantation dose will be given to thepatient about 5 days prior to bone marrow transplantation. Additional,earlier doses 6, 7, or 8 days prior to bone marrow transplantation canalso be given. It may be desirable to administer a first treatment, thento repeat pre-bone marrow administrations every 1-5 days. A blocker canalso be administered one, two, three, or more times after donor bonemarrow transplantation. Typically, a post-bone marrow transplanttreatment will be given about 2-14 days after bone marrowtransplantation. The post-bone marrow administration can be repeated asmany times as needed. If more than one administration is given theadministrations can be spaced about 1 week apart. Additional doses canbe given if the patient appears to undergo early or unwanted T cellrecovery. Preferably, a blocker is administered at least once (andpreferably two, three, or more times) prior to donor bone marrowtransplantation and at least once (and preferably two, three, or moretimes) after donor bone marrow transplantation.

While not wishing to be bound by theory the inventor believes thatrepeated stem cell administration may promote chimerism and possiblylong-term deletional tolerance in graft recipients. Accordingly, anymethod referred to herein which includes the administration ofhematopoietic stem cells can further include multiple administrations ofstem cells. In preferred embodiments: a first and a secondadministration of stem cells are provided prior to the implantation of agraft; a first administration of stem cells is provided prior to theimplantation of a graft and a second administration of stem cells isprovided at the time of implantation of the graft. In other preferredembodiments: a first administration of stem cells is provided prior toor at the time of implantation of a graft and a second administration ofstem cells is provided subsequent to the implantation of a graft. Theperiod between administrations of hematopoietic stem cells can bevaried. In preferred embodiments a subsequent administration ofhematopoietic stem cell is provided: at least two days, one week, onemonth, or six months after the previous administration of stem cells; atleast two days, one week, one month, or six months after theimplantation of the graft.

The method can further include the step of administering a second orsubsequent dose of hematopoietic stem cells: when the recipient beginsto show signs of rejection, e.g., as evidenced by a decline in functionof the grafted organ, by a change in the host donor specific antibodyresponse, or by a change in the host lymphocyte response to donorantigen; when the level of chimerism decreases; when the level ofchimerism falls below a predetermined value; when the level of chimerismreaches or falls below a level where staining with a monoclonal antibodyspecific for a donor PBMC antigen is equal to or falls below stainingwith an isotype control which does not bind to PBMC's, e.g. when thedonor specific monoclonal stains less than 1-2% of the cells; orgenerally, as is needed to maintain tolerance or otherwise prolong theacceptance of a graft. Thus, method of the invention can be modified toinclude a further step of determining if a subject which has received aone or more administrations of hematopoietic stem cells is in need of asubsequent administration of hematopoietic stem cells, and if so,administering a subsequent dose of hematopoietic stem cells to therecipient.

Other embodiments are within the following claims.

1-24. (canceled)
 25. A method of preparing a human recipient for a graftfrom a human donor, without administering irradiation to promote theformation of mixed chimerism and without thymic irradiation, the methodcomprising: administering to the recipient an anti-CD2 monoclonalantibody; administering to the recipient, donor peripheral bloodprogenitor in an amount sufficient to form mixed chimerism in theabsence whole body irradiation and thymic irradiation; administering tothe recipient, a non-chronic course of an immunosuppressant; andadministering to the recipient, one or both of a blocker of the CD40ligand-CD40 interaction and a blocker of the CD28-B7 interaction. 26.The method of claim 25, wherein an anti-CD2 monoclonal antibody isadministered on days −4, −3, −2, −1.
 27. The method of claim 25, whereinthe method further includes, prior to one or more administration of ananti-CD2 antibody, administering to the recipient a steroid.
 28. Themethod of claim 25, wherein the non-chronic course is 35-day course ofcyclosporine followed by 7 days of tapering and ending on day
 42. 29.The method of claim 25, further comprising introducing the graft intothe recipient.
 30. The method of claim 25, wherein one or both of ananti-CD40L antibody and CTLA4/Ig are administered to the recipient. 31.The method of claim 25, wherein the method includes: administering ananti-CD2 monoclonal antibody on days −4, −3, −2, −1; performing asplenectomy on the recipient on the day of transplantation;administering to the recipient donor peripheral blood progenitor cellson the day of transplantation; administering to the recipient 35-daycourse of cyclosporine followed by 7 days of tapering and ending on day42; and administering to the recipient one or both of an anti-CD40Lantibody and CTLA4/Ig.
 32. The method of claim 25, further comprisingperforming a splenectomy on the recipient.